Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (“PrP”) [Bolton et al., Science 218:1309–11 (1982); Prusiner et al., Biochemistry 21:6942–50 (1982); McKinley et al., Cell 35:57–62 (1983)]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene [Basler et al., Cell 46:417–28 (1986)] and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrPC into a modified form called PrPSc.
At present, it appears that the scrapie isoform of the prion protein (PrPSc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S. B., “Molecular biology of prion disease,” Science 252:1515–1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. and Wells, Microbiol. Immunol. 172:21–38 (1991)]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D. C., Science 197:943–960 (1977); Medori et al., N. Engl. J. Med. 326:444–449 (1992)]. The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Some cases of human prion disease have been transmitted to rodents but apparently with less regularity than transmission between animals of the same species [Gibbs, Jr. et al., Slow Transmissible Diseases of the Nervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow,. eds. (New York: Academic Press), pp. 87–110 (1979); Tateishi et al., Prion Diseases of Humans and Animals, Prusiner et al., eds. (London: Ellis Horwood), pp. 129–134 (1992)]. The infrequent transmission of human prion disease to rodents has been cited as an example of the “species barrier” first described by Pattison in his studies of passaging the scrapie agent between sheep and rodents [Pattison, I. H., NINDB Monograph 2, D. C. Gajdusek, C. J. Gibbs Jr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing), pp. 249–257 (1965)]. In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only a few animals developing illness. Subsequent passage in the same species was characterized by all the animals becoming ill after greatly shortened incubation times.
The molecular basis for the species barrier between Syrian hamster (SHa) and mouse was shown to reside in the sequence of the PrP gene using transgenic (Tg) mice [Scott et al., Cell 59:847–857 (1989)]. SHaPrP differs from MoPrP at 16 positions out of 254 amino acid residues [Basler et al., Cell 46:417–428 (1986); Locht et al., Proc. Natl. Acad. Sci. USA 83:6372–6376 (1986)]. Tg(SHaPrP) mice expressing SHaPrP had abbreviated incubation times when inoculated with SHa prions. When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long. Thus, it has not been possible, for example in the case of human prions, to use transgenic animals (such as mice containing a PrP gene of another species) to reliably test a sample to determine if that sample is infected with prions. Such a test was first disclosed in application Ser. No. 08/242,188 filed May 13, 1994 which is now U.S. Pat. No. 5,565,186 issued Oct. 15, 1996.
Most human CJD cases are sporadic, but about 10–15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene [Hsiao et al., Neurology 40:1820–1827 (1990); Goldfarb et al., Science 258:806–808 (1992); Kitamoto et al., Proc. R. Soc. Lond. 343:391–398 (1994)]. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown et al., Lancet 340:24–27 (1992)]. Despite numerous attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date [Harries-Jones et al., J. Neurol. Neurosurg. Psychiatry 51:1113–1119 (1988)] except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases of the Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp.66–90 (1979)].
More than 45 young adults previously treated with HGH derived from human pituitaries have developed CJD [Koch et al., N. Engl. J. Med. 313:731–733 (1985); Brown et al., Lancet 340:24–27 (1992); Fradkin et al., JAMA 265:880–884 (1991); Buchanan et al., Br. Med. J. 302:824–828 (1991)]. Fortunately, recombinant HGH is now used, although the seemingly remote possibility has been raised that increased expression of wt PrPC stimulated by high HGH might induce prion disease [Lasmezas et al., Biochem. Biophys. Res. Commun. 196:1163–1169 (1993)]. That the HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH [Gibbs, Jr. et al., N. Engl. J. Med. 328:358–359 (1993)]. The long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD in thousands of people treated with HGH worldwide. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotrophin hormone [Healy et al., Br. J. Med. 307:517–518 (1993); Cochius et al., Aust. N.Z. J. Med. 20:592–593 (1990); Cochius et al., J. Neurol. Neurosurg. Psychiatry 55:1094–1095 (1992)] as well as at least 11 patients receiving dura mater grafts [Nisbet et al., J. Am. Med. Assoc. 261:1118 (1989); Thadani et al., J. Neurosurg. 69:766–769 (1988); Willison et al., J. Neurosurg. Psychiatric 54:940 (1991); Brown et al., Lancet 340:24–27 (1992)]. These cases of iatrogenic CJD underscore the need for screening pharmaceuticals that might possibly be contaminated with prions.
Two doctors in France were charged with involuntary manslaughter of a child who had been treated with growth hormones extracted from corpses. The child developed Creutzfeldt-Jakob Disease. (See New Scientist, Jul. 31, 1993, page 4). According to the Pasteur Institute, since 1989 there have been 24 reported cases of CJD in young people who were treated with human growth hormone between 1983 and mid-1985. Fifteen of these children have died. It now appears as though hundreds of children in France have been treated with growth hormone extracted from dead bodies at the risk of developing CJD (see New Scientist, Nov. 20, 1993, page. 10.) In view of such, there clearly is a need for a convenient, cost-effective method for producing human products such as growth hormone that are free from any potentially contagious prion contamination.
The risk of transmitting prion-related disorders through therapeutic human products is a serious health concern. One method for preventing the transmission of prion related disorders is to produce recombinant human products in organisms such as Escherichia coli and Saccharomyces cerevisiae, since these organisms do not have an endogenous PrP gene and thus are not susceptible to PrPSc infection While Escherichia coli and Saccharomyces cerevisiae production is ideal for the large scale synthesis of many human proteins, factors such as plasmid stability and insolubility of the desired protein product may limit the usefulness of these systems in some circumstances. In addition, certain recombinantly-produced proteins require post-translational modification to obtain the function of the endogenous protein, and thus may require synthesis in mammalian cells or even species-specific cell lines for proper functioning of the produced protein. For example, a recombinant human thryotropin (rhTSH) produced in Chinese Hamster Ovary cells is more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. The rhTSH also has a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH compared to pituitary hTSH. (Thotakura et al., Endocrinology 128:341–348 (1991)) Since it is desirable to use therapeutic agents with the proper post-translational modifications, mammalian systems are preferable for the production of such proteins.
Moreover, other therapeutic agents, such as antibodies, are exclusively produced by mammalian cell systems. Classical cell fusion techniques allow efficient production of monoclonal antibodies by fusing the B cell producing the antibody with an immortalized mammalian cell line. The resulting cell line is called a hybridoma cell line. Applications of human antibodies produced by these hybridoma systems have promising potential in the area of cancer, immunodeficiencies, and other diseases involving an immune response. For instance, the apoptosis-inducing human monoclonal antibody SC-1 has been shown to cause a significant induction of apoptotic activity in eight patients with poorly differentiated stomach adenocarcinoma (Vollmers et al. Oncol Rep 5:549–552 (1998)). In another example, the antibody to HER2/neu has been shown to be a promising therapy for human breast cancer (Valero, (1998) Semin. Oncol. 5: 549–552). Monoclonal antibodies produced in murine hybridoma systems require an additional step of “humanizing” the antibodies to prevent the antibodies from being recognized as foreign epitopes (See e.g. Sato et al., (1994) Mol. Immunol. 31: 371–381). These systems are susceptible to prion infection, and antibodies produced in infected cells pose a risk of transmission to any individual receiving antibodies from the infected sources.
Since many therapeutics are produced in mammalian systems, there is a need for ensuring the safety of the products isolated from such systems. Given the potential for the transmission of disease when these therapeutics are extracted from tissue, there is a need for a method of producing therapeutics that are free from the risk of human disease-causing contaminants such as prions.